Characterization of a Novel Capsid Assembly Modulator for the Treatment of Chronic Hepatitis B Virus Infection

ABSTRACT The standard of care for the treatment of chronic hepatitis B (CHB) is typically lifelong treatment with nucleos(t)ide analogs (NAs), which suppress viral replication and provide long-term clinical benefits. However, infectious virus can still be detected in patients who are virally suppressed on NA therapy, which may contribute to the failure of these agents to cure most CHB patients. Accordingly, new antiviral treatment options are being developed to enhance the suppression of hepatitis B virus (HBV) replication in combination with NAs (“antiviral intensification”). Here, we describe GS-SBA-1, a capsid assembly modulator (CAM) belonging to class CAM-E, that demonstrates potent inhibition of extracellular HBV DNA in vitro (EC50 [50% effective concentration] = 19 nM) in HBV-infected primary human hepatocytes (PHHs) as well as in vivo in an HBV-infected immunodeficient mouse model. GS-SBA-1 has comparable activities across HBV genotypes and nucleos(t)ide-resistant mutants in HBV-infected PHHs. In addition, GS-SBA-1 demonstrated in vitro additivity in combination with tenofovir alafenamide (TAF). The administration of GS-SBA-1 to PHHs at the time of infection prevents covalently closed circular DNA (cccDNA) formation and, hence, decreases HBV RNA and antigen levels (EC50 = 80 to 200 nM). Furthermore, GS-SBA-1 prevents the production of extracellular HBV RNA-containing viral particles in vitro. Collectively, these data demonstrate that GS-SBA-1 is a potent CAM that has the potential to enhance viral suppression in combination with an NA.


Differential Scanning Fluorimetry
Capsid particles were assembled by addition of 500 mM NaCl to a solution of 40 μM of Cp149 dimers in buffer containing 25 mM Hepes, pH 7.5, and 2.5 mM DTT, followed by incubation for 2 hours at room Protein and compound were pre-incubated for 1 hour at room temperature in buffer containing 50 mM Hepes, pH 7.5, 2.5 mM DTT, and 100 mM NaCl or 500 mM NaCl for studies with Cp149 dimer or assembled capsid, respectively. SYPRO Orange was added at 1:1000 (v/v) dilution. All concentrations were final after mixing. Thermal denaturation was performed by increasing the temperature from 20°C to 99.5°C at 1°C/minute on a ViiA7 Real-Time PCR instrument (Life Technologies, Carlsbad, CA). Fluorescence intensity data were collected at intervals of 0.07°C. The assay was performed in the absence and presence of compounds. Fluorescence intensity data were analyzed with Protein Thermal Shift Software (Life Technologies, Carlsbad, CA) using a first derivative approach to calculate Tm. The change in protein melting temperature (ΔTm) was calculated by subtracting the Tm value obtained for protein in the presence of 1% DMSO (no compound) from the Tm value obtained in the presence of a compound and 1% DMSO.

HepAD38 Virion Production
Supernatant containing virions from HepAD38 cells cultured in the absence of tetracycline was collected every 3 to 4 days and virions were precipitated with PEGit (Systems Biosciences, Palo Alto, CA; LV825A-1) overnight at 4°C. Following precipitation, supernatant was spun at 3000 rpm at 4°C for 15 minutes and the pellet containing the virions was resuspended in William's E medium (Thermo Fisher Scientific, A1217601) containing 25% fetal bovine serum (Thermo Fisher Scientific, 10082147). The viral titer was determined by quantitative PCR (qPCR).
Approximately 24 hours after plating, PHH were infected with HepAD38-derived HBV virions (4) (genotype D virus) at 500 viral genome equivalents per cell in maintenance medium supplemented with 4% PEG 8000 (Promega, Madison, WI; V3011). The infection was allowed to proceed for 20 -24 hours before removing remaining extracellular virions by washing with maintenance medium 3 times.

HepG2-NTCP Culture Conditions and Bulk Infection
HepG2-NTCP cells were seeded in T175 flasks 3 days prior to infection in basic growth medium (DMEM medium (Thermo Fisher Scientific, 11965-092), 200mM, Thermo Fisher Scientific, 25030-081)) containing On Day 4, cells were removed from the flask using 0.1% trypsin (Thermo Fisher Scientific, 15400054) and trypsinization was halted by adding basic growth medium. Cells were centrifuged at 1200 rpm for 5 minutes (Beckman Coulter, Allegra X-14R). Cells were subjected to 2 additional wash steps with OptiMEM and resuspended after the last wash in antiviral assay medium (basic growth media containing 2% FBS and 1% DMSO) at 0.2 x 10 6 cells/mL. Cells were plated in collagen coated 96-well plates (Thermo Fisher Scientific; CM1096) at a density of 30,000 cells per well and treated with compounds.

Compound Treatment
Three or four days following infection of PHH or HepG2-NTCP cells, respectively, with AD38 virus (Day 100% DMSO using the HP Digital Dispenser D300 (Hewlett Packard, Palo Alto, CA). HBV-infected PHH and HepG2-NTCP cells were dosed with compounds using serial dilutions ranging from 2 μM to 0.31 nM.
DMSO was used as the vehicle control. Vehicle and compound containing wells were normalized such that the final concentration of DMSO was 1.7% (v/v). Medium containing compound was removed and PHH or HepG2-NTCP cells were re-dosed with new media containing the appropriate compounds on Day 3 following the initial dose for a total of 2 doses. On Day 6 post-initiation of dosing for both PHH and HepG2-NTCP culture systems, supernatant and cells collected to assess antiviral activity and cytotoxicity.

Compound Treatment Assays at the Time of HBV Infection in PHH
HepAD38-derived HBV virions were dosed with compounds using serial dilutions ranging from 2 μM to 0.31 nM and incubated for 1 hour at room temperature. Approximately 24 hours after plating in collagen coated 96-well plates (Thermo Fisher Scientific; CM1096) at a density of 55,000 to 75,000 live PHH cells per well, medium was removed and HepAD38-derived HBV virions in the presence of compounds were transferred from v-shaped 96-well plates to PHH-containing 96-well plates. The infection was allowed to proceed for 20 -24 hours in triplicate wells for each compound concentration before removing remaining extracellular virions by washing with William's E medium 3 times. Maintenance medium was replenished, and cells were dosed with compound supplied in 100% DMSO using the HP Digital Dispenser D300.
Maintenance media with tested compounds were replenished for total of 3 times on Days 1, 3, and 6 after the infection.

Extracellular hepatitis B virus S antigen (HBsAg) and hepatitis B virus E antigen (HBeAg) were detected
in culture media by an electrochemiluminescence assay (MSD) as previously reported (5).

Quantification of Extracellular DNA by qPCR
Viral DNA from PHH supernatants was purified using the Qiagen DNeasy 96 kit (69582) following the manufacturer's recommended protocol. Quantification of vDNA by qPCR (quantitative polymerase chain reaction) amplification of the HBx region of the genome was performed by combining 5 μL of DNA, 900 nM of HBx forward primer, 900 nM of reverse primer (Supplemental Table 3), 0.2 μM TaqMan probe, and 1x TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific; 4444557) for a total reaction volume of 20 μL in 96-well PCR plates (Thermo Fisher Scientific; 4346906). qPCR was carried out on a real-time PCR system (Thermo Fisher Scientific; QuantStudio 7 Flex) using the following conditions: 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. A plasmid containing the HBV full genome was used for the standard curve.

Quantification of Cytotoxicity
Cytotoxicity was measured by alamarBlue® (Thermo Fisher Scientific, DAL1100). Following removal of all culture supernatant, cells were replenished with maintenance (PHH) or antiviral assay (HepG2-NTCP) medium containing 10% (v/v) alamarBlue® dye. Cells were incubated at 37ºC for 3 hours. Fluorescence was measured using a fluorescence excitation wavelength of 540 nm and fluorescence emission was read at 585 nm on a spectrophotometer (Molecular Devices, SpectraMax M5) and analyzed with SoftMax Pro 6.3 software (Molecular Devices).

Quantification of Intracellular Viral RNA
Intracellular HBV viral RNA (vRNA) was isolated from PHH using the RNeasy 96 kit (Qiagen, 74182) following the manufacturer's recommended protocol. Quantification of vRNA by qRT-PCR (quantitative reverse transcription polymerase chain reaction) amplification of the HBVX region of the genome was performed by combining 5 μL of RNA to 900 nM of HBVX forward primer, 900 nM of HBVX reverse primer (Supplement Table 4), 0.2 μM TaqMan probe (Supplemental Table 4), and 1x beta-actin (ACTB) endogenous transcripts (Thermo Fisher Scientific; 4310881E) and 1x TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific; 4444434) for a total reaction volume of 20 μL in 96-well PCR plates (Thermo Fisher Scientific; 4346906). qRT-PCR was carried out on a real-time PCR system (Thermo Fisher Scientific; QuantStudio 7 Flex) using the following conditions: 50°C for 5 minutes, then 95°C for 20 seconds, followed by 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds. ACTB mRNA expression was used to normalize target gene expression. Levels of HBV mRNA for all donors were calculated as fold change relative to no drug treated sample using the 2-ΔΔCt method (6).

Data Analysis
Antiviral activity or cytotoxicity of each test compound was determined from vRNA, vDNA, HBeAg, HBsAg, and alamarBlue® data by comparing compound-treated PHH to DMSO-treated PHH to generate a percent of DMSO control value (% DMSO control). The % DMSO control was calculated by the

HBV Cellular DNA Extraction and Purification
Extraction of HBV cellular DNA was carried out using precipitation procedure which was described in detail by manufacturer's instruction (EPICENTRE; MC85200) with minor modifications. Briefly, cells in 24-well plate were washed twice with 1× PBS and lysed in 0.5 mL of Tissue and Cell Lysis Solution. After 10 minutes incubation at room temperature, the lysate was transferred into a 1 mL tube, followed by the addition of 0.25 mL of MPC Protein Precipitation Reagent. After 10 minutes on ice, the lysate was clarified by centrifugation (Eppendorf; Centrifuge 5430R) at ≥10,000 × g for 10 minutes at 4°C. The supernatant was transferred to a clean microcentrifuge tube and DNA extracted with 0.75 mL of isopropanol by centrifugation at ≥10,000 × g for 10 minutes at 4°C. HBV cellular DNA pellet was rinsed twice with 1 mL of 70% ethanol and dissolved in 100 μL of TE buffer. Extracted HBV cellular DNA was purified using Zymo-Spin™ IIC Column which was described in detail by manufacturer's instruction (ZYMO Research; D4034). Briefly, 100 μL of HBV cccDNA was mixed with 200 μL of DNA Binding Buffer, transferred into the Zymo-Spin™ IIC column in a collection tube, and centrifugation (Eppendorf; Centrifuge 5415D) at ≥10,000 × g for 30 seconds. HBV cccDNA bound to the membrane was washed twice with 300 μL of DNA Wash Buffer. After washing, the column was transferred to a clean 1.5 mL microcentrifuge tube.
HBV cccDNA was eluted with 30 μL of DNA Elution Buffer.

HBV cccDNA Quantitation by qPCR
HBV cellular DNA was treated with T5 exonuclease (New England Biolabs; M0363S) according to manufacturer's instructions prior to quantification by qPCR. HBV cccDNA was quantified by real-time PCR using 1× TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific; 4444557) on real-time PCR system (Thermo Fisher Scientific; QuantStudio 7 Flex). PCR was carried out in the following conditions: initial denaturation for 20 seconds at 95°C, followed by 40 cycles at 95°C denaturation for 1 second and annealing/elongation at 60°C for 20 seconds. cccDNA specific primers which targeted DR1-DR2 gap region (Supplemental Table 4) were used throughout this study. HBV cccDNA copy number was quantified relative to a linearized plasmid DNA standard.

Southern Blotting Analysis
Purified HBV cellular DNA samples were resolved in a 1.2% agarose gel (Roche; 11388983001) in 1× TAE buffer (TEKnova; T1260). After electrophoresis, the DNA was depurinated, denatured (Biosciences; R016), and neutralized (Biosciences; R018) exactly as described by Cai et al. (7) and transferred onto Nytran SuPerCharge membrane (GE Healthcare, 10416216) using the TurboBlotter system (GE Healthcare; 10416300). The membrane was hybridized with an HBV-DNA probe by branched DNA signal amplification (bDNA) method. All reagents for the bDNA signal amplification method were from Affymetrix. Briefly, transfer membranes were pre-hybridized with lysis mixture (cat# QG0504) and blocking reagent (cat#QS0505) at 55°C for 30 minutes. After, HBV DNA target probe, QG (cat# QS1051), was added and allowed to hybridize overnight at 55°C. The next day, pre-Amplifier (2.0 PreAmp cat# QG15905), amplifier (2.0 Amp cat# QG15098) and label probe containing alkaline phosphatase (2.0 Label Probe cat#QG1324) were added sequentially. Pre-amplifier and amplifier mixtures were incubated at 55°C for 1 hour while the label probe was incubated at 50°C for 1 hour. Between each incubation step the membrane was washed 3 times with QuantiGene Wash Buffer (cat# QG0509). Following bDNA hybridization procedures, HBV DNA was detected using CDP-Star (GE Healthcare; NIF1229) according to manufacturer's instructions. Images were acquired with ImageQuant LAS 4000 (GE Healthcare) with exposure for a few second to minutes. Density of cccDNA bands was determined using ImageQuant TL software (version 7, GE Healthcare).

RNA-Seq analysis
RNA-Seq was conducted by Q2 Expression Analysis (Durham, NC) as described previously (5). Briefly, extracellular RNA was isolated using TRizol (Thermo Fisher Scientific) from HepAD38 cells supernatants treated with DMSO control or compounds at 50X EC50. cDNA libraries were constructed using a TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA). Pair-end sequencing was conducted using Illumina HiSeq2000 with read length of 50 nucleotides. On average, approximately 30 million reads were generated per sample. Sequencing reads were aligned to the human and HBV genomes by STAR method (8). RNASeq data were deposited in SRA with BioProject accession PRJNA906647 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA906647).

Pharmacokinetic Studies
To evaluate pharmacokinetics of GS-SBA-1 at steady state, uninfected uPA-SCID mice were treated orally with 5 mg-eq/kg TAF or 100 mg-eg/kg of GS-SBA-1P, a prodrug of GS-SBA-1 once daily for 14 days.

uPA-SCID Mouse Model
Male uPA/SCID mice between 12-18 weeks of age with humanized liver (cDNA-uPA wild/+ /SCID [cDNA-uPA wild/+ : B6;129SvEv-Plau, SCID:C.B-17/Icr-scid /scid Jcl) were produced as previously described by PhoenixBio, Co. Ltd. (Japan) (11). Briefly, frozen human hepatocytes (donor BD195, Corning Incorporated, Tewksbury, MA, USA) were thawed and transplanted into 2-to 4-week-old uPA/SCID mice by splenic injection. Mice were selected for studies if their liver reconstitution levels had an estimated replacement index greater than 70% based on the blood concentration of human albumin (>8.5mg/mL) one week prior to study initiation. General health observations including weight were monitored weekly. All animal protocols were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Welfare Committee of Phoenix Bio Co., Ltd. All mice were housed individually and maintained in accordance with the Animal Ethics Committee of PhoenixBio (resolution #2214).
HBV-infected mice were randomized into three different treatment groups based on body weight, blood h-Alb, and serum HBV DNA concentrations. All mice had blood h-Alb levels above 12 mg/mL and serum HBV DNA levels above 8.8x10 8 copies/mL. Mice received an oral dose of 100 mg-eq/kg of GS-SBA-1P, 5 mg-eq/kg of TAF or dosing vehicle (vehicle control) once daily for 84 days. GS-SBA-1P was dosed in 1% (w/w) HPMC and 0.3% (w/w) polysorbate 80 in water, while TAF was dosed in 10 mM phosphate buffer, pH 6.5. Mice were followed for an additional 28 days following cessation of treatment at day 84.
Mouse serum was monitored weekly for serum HBV DNA and HBV antigens.

Intracellular Metabolites of TAF
Primary human hepatocytes were purchased from Life Technologies (Grand Island, NY) and BioreclamationIVT (Baltimore, MD) were plated in collagen-coated 12-well tissue culture plates with LC-MS/MS as previously described (12,13).

Determination of TFV, TFV-MP, and TFV-DP in Primary Human Hepatocytes
Analytes were separated using a 50 x 2 mm x 2.  The assay schematic is depicted in Figure 2A. a EC 50 calculated by 4-parameter logistic curve fitting data from 6-day treatment of HBV-infected PHH. The top of the curve was constrained to 100. Averages ± SD are shown for four independent experiments for donor A and averages of two independent experiments for donors B through D are shown. b 6-day treatment of HBV-infected HepG2-NTCP cells. The top of the curve was constrained to 100. Averages ± SD are shown across four independent experiments for GS-SBA-1 and average of two independent experiments for TFV are shown. *HepG2-NTCP cells were treated with TAF Supplemental Table 2. Antiviral activity of GS-SBA-1 in PHH treated at the time of HBV infection The assay schematic is depicted in Figure 3A. a Calculated by 4-parameter logistic curve fitting of data from 6-day treatment of HBV-infected PHH. The top was constrained to 100%. Averages of two independent experiments are shown.